Subtilisin-like Serine Protease Gene Research Articles

Genome-wide identification and analysis of Subtilisin-like serine protease gene family in banana (Musa accuminta L.) and their expression under abiotic stresses

Genome-wide identification and analysis of Subtilisin-like serine protease gene family in banana (Musa accuminta L.) and their expression under abiotic stresses

Metarhizium robertsii ammonium permeases (MepC and Mep2) contribute to rhizoplanecolonization and modulates the transfer of insect derived nitrogen to plants.

The endophytic insect pathogenic fungi (EIPF) Metarhizium promotes plant growth through symbiotic association and the transfer of insect-derived nitrogen. However, little is known about the genes involved in this association and the transfer of nitrogen. In this study, we assessed the involvement of six Metarhizium robertsii genes in endophytic, rhizoplane and rhizospheric colonization with barley roots. Two ammonium permeases (MepC and Mep2) and a urease, were selected since homologous genes in arbuscular mycorrhizal fungi were reported to play a pivotal role in nitrogen mobilization during plant root colonization. Three other genes were selected on the basis on RNA-Seq data that showed high expression levels on bean roots, and these encoded a hydrophobin (Hyd3), a subtilisin-like serine protease (Pr1A) and a hypothetical protein. The root colonization assays revealed that the deletion of urease, hydrophobin, subtilisin-like serine protease and hypothetical protein genes had no impact on endophytic, rhizoplane and rhizospheric colonization at 10 or 20 days. However, the deletion of MepC resulted in significantly increased rhizoplane colonization at 10 days whereas ΔMep2 showed increased rhizoplane colonization at 20 days. In addition, the nitrogen transporter mutants also showed significantly higher 15N incorporation of insect derived nitrogen in barley leaves in the presence of nutrients. Insect pathogenesis assay revealed that disruption of MepC, Mep2, urease did not reduce virulence toward insects. The enhanced rhizoplane colonization of ΔMep2 and ΔMepC and insect derived nitrogen transfer to plant hosts suggests the role of MepC and Mep2 in Metarhizium-plant symbiosis.

Cell remodeling and subtilase gene expression in the actinorhizal plant Discaria trinervis highlight host orchestration of intercellular Frankia colonization.

Nitrogen-fixing filamentous Frankia colonize the root tissues of its actinorhizal host Discaria trinervis via an exclusively intercellular pathway. Here we present studies aimed at uncovering mechanisms associated with this little-researched mode of root entry, and in particular the extent to which the host plant is an active partner during this process. Detailed characterization of the expression patterns of infection-associated actinorhizal host genes has provided valuable tools to identify intercellular infection sites, thus allowing invivo confocal microscopic studies of the early stages of Frankia colonization. The subtilisin-like serine protease gene Dt12, as well as its Casuarina glauca homolog Cg12, are specifically expressed at sites of Frankia intercellular colonization of D.trinervis outer root tissues. This is accompanied by nucleo-cytoplasmic reorganization in the adjacent host cells and major remodeling of the intercellular apoplastic compartment. These findings lead us to propose that the actinorhizal host plays a major role in modifying both the size and composition of the intercellular apoplast in order to accommodate the filamentous microsymbiont. The implications of these findings are discussed in the light of the analogies that can be made with the orchestrating role of host legumes during intracellular root hair colonization by nitrogen-fixing rhizobia.

Subtilisin-like serine protease gene TghSS42 from Trichoderma ghanense ACCC 30153 was successfully expressed in Escherichia coli and recombinant protease rTghSS42 exhibited antifungal ability to five phytopathogens.

The subtilisin-like serine protease gene TghSS42 was cloned from biocontrol agent Trichoderma ghanense ACCC 30153. Its coding region is 1302 bp in length, encoding 433 aa with a predicted protein molecular weight of 42.5 kDa and pI of 5.53. The accession number of cDNA sequence of TghSS42 gene is KJ740359. Furthermore, the transcription of the TghSS42 gene was all up-regulated under nine different treatments by RT-qPCR analysis, and the highest transcription level of TghSS42 approached 177.29-fold at 4 h under induction with 1% (w/v) Alternaria alternata cell walls, indicating that TghSS42 could be induced by the plant or phytopathogen. Furthermore, Escherichia coli recombinant strain BL21-TghSS42 was constructed. The recombinant protease rTghSS42, with an expected molecular weight of approximately 68.5 kDa (containing 26.0 kDa GST tag), has been successfully expressed and purified from BL21-TghSS42. The purified protease rTghSS42 activity reached a peak of 18.7 U/mL at 4 h following 1.0 mM IPTG induction. The optimal enzyme reaction temperature was 40℃ and the optimal pH was 7.0. The recombinant protease rTghSS42 exerted broad-spectrum antifungal ability against Rhizoctonia solani, Fusarium oxysporum, A. alternata, Sclerotinia sclerotiorum and Cytospora chrysosperma. The inhibition rate of mycelial growth varied between 21.2% and 50.0%.

Molecular cloning and characterization of three novel subtilisin-like serine protease genes from Hevea brasiliensis

Subtilisin-like serine proteases play important roles in a variety of biological functions. In this study, three novel subtilisin-like serine protease genes (HbSPA, HbSPB, and HbSPC) were isolated from Hevea brasiliensis leaves treated with salicylic acid using RT-PCR followed by RACE. HbSPA, HbSPB and HbSPC genes encode polypeptides consisting of 766, 748, and 748 amino acids, respectively, which belong to members of the subtilisin-like serine protease family from plants. Semi-qRT-PCR revealed that HbSPA was expressed in Hevea leaf, stem, hypocotyl, and root, whereas HbSPB and HbSPC were also expressed in the latex. qRT-PCR demonstrated that the expression of HbSPA was significantly induced during P. palmivora infection and slightly induced by wounding, while the expression of HbSPB or HbSPC was notably suppressed during P. palmivora infection and highly induced by salicylic acid treatment. Our results implied that HbSPA might be involved in rubber tree defense mechanisms against P. palmivora.

Whole genome annotation and comparative genomic analyses of bio-control fungus Purpureocillium lilacinum.

BackgroundThe fungus Purpureocillium lilacinum is widely known as a biological control agent against plant parasitic nematodes. This research article consists of genomic annotation of the first draft of whole genome sequence of P. lilacinum. The study aims to decipher the putative genetic components of the fungus involved in nematode pathogenesis by performing comparative genomic analysis with nine closely related fungal species in Hypocreales.Resultsde novo genomic assembly was done and a total of 301 scaffolds were constructed for P. lilacinum genomic DNA. By employing structural genome prediction models, 13, 266 genes coding for proteins were predicted in the genome. Approximately 73 % of the predicted genes were functionally annotated using Blastp, InterProScan and Gene Ontology. A 14.7 % fraction of the predicted genes shared significant homology with genes in the Pathogen Host Interactions (PHI) database. The phylogenomic analysis carried out using maximum likelihood RAxML algorithm provided insight into the evolutionary relationship of P. lilacinum. In congruence with other closely related species in the Hypocreales namely, Metarhizium spp., Pochonia chlamydosporia, Cordyceps militaris, Trichoderma reesei and Fusarium spp., P. lilacinum has large gene sets coding for G-protein coupled receptors (GPCRs), proteases, glycoside hydrolases and carbohydrate esterases that are required for degradation of nematode-egg shell components. Screening of the genome by Antibiotics & Secondary Metabolite Analysis Shell (AntiSMASH) pipeline indicated that the genome potentially codes for a variety of secondary metabolites, possibly required for adaptation to heterogeneous lifestyles reported for P. lilacinum. Significant up-regulation of subtilisin-like serine protease genes in presence of nematode eggs in quantitative real-time analyses suggested potential role of serine proteases in nematode pathogenesis.ConclusionsThe data offer a better understanding of Purpureocillium lilacinum genome and will enhance our understanding on the molecular mechanism involved in nematophagy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2229-2) contains supplementary material, which is available to authorized users.

Functional analysis of a subtilisin-like serine protease gene from biocontrol fungus Trichoderma harzianum

The subtilisin-like serine protease gene ThSS45 has been cloned from Trichoderma harzianum ACCC30371. Its coding region is 1302 bp in length, encoding 433 amino acids, with a predicted protein molecular weight of 44.9 kDa and pI of 5.91. ThSS45 was shown by RT-qPCR analysis to be differentially transcribed in response to eight different treatments. The transcription of ThSS45 was up-regulated when grown in mineral medium, under carbon starvation, and nitrogen starvation, and in the presence of 1% root powder, 1% stem powder, and 1% leaf powder derived from Populus davidiana × P. bolleana (Shanxin poplar) aseptic seedlings. The highest increase in transcription approached 3.5-fold that of the control at 6 h under induction with 1% poplar root powder. The transcription of ThSS45 was also slightly up-regulated by 1% Alternaria alternata cell wall and 5% A. alternata fermentation liquid. Moreover, the analyses of coding and promoter regions of ThSS45 homologs indicated that serine protease may be involved in both mycoparasitism and antibiotic secretion. ThSS45 was cloned into the pGEX-4T-2 vector and then expressed in Escherichia coli BL21. The recombinant protein, with an expected molecular weight of approximately 69 kDa, was then purified. When transformant BL21-ss was induced with 1 mM IPTG for 6 h, the purified protease activity reached a peak of 18.25 U/ml at pH 7.0 and 40°C. In antifungal assays the purified protease obviously inhibited the growth of A. alternata mycelia.

Truncated Cotton Subtilase Promoter Directs Guard Cell-Specific Expression of Foreign Genes in Tobacco and Arabidopsis

A 993-bp regulatory region upstream of the translation start codon of subtilisin-like serine protease gene was isolated from Gossypium barbadense. This (T/A)AAAG-rich region, GbSLSP, and its 5′- and 3′-truncated versions were transferred into tobacco and Arabidopsis after fusing with GUS or GFP. Histochemical and quantitative GUS analysis and confocal GFP fluorescence scanning in the transgenic plants showed that the GbSLSP-driven GUS and GFP expressed preferentially in guard cells, whereas driven by GbSLSPF2 to GbSLSPF4, the 5′-truncated GbSLSP versions with progressively reduced Dof1 elements, both GUS and GFP expressed exclusively in guard cells, and the expression strength declined with (T/A)AAAG copy decrement. Deletion of 5′-untranslated region from GbSLSP markedly weakened the activity of GUS and GFP, while deletion from the strongest guard cell-specific promoter, GbSLSPF2, not only significantly decreased the expression strength, but also completely abolished the guard cell specificity. These results suggested both guard cell specificity and expression strength of the promoters be coordinately controlled by 5′-untranslated region and a cluster of at least 3 (T/A)AAAG elements within a region of about 100 bp relative to transcription start site. Our guard cell-specific promoters will enrich tools to manipulate gene expression in guard cells for scientific research and crop improvement.

Characterization, cloning, and heterologous expression of a subtilisin-like serine protease gene VlPr1 from Verticillium lecanii

The entomopathogenic fungus Verticillium lecanii is a well-known biocontrol agent. V. lecanii produces subtilisin-like serine protease (Pr1), which is important in the biological control activity of some insect pests by degrading insect cuticles. In this study, a subtilisin-like serine protease gene VlPr1 was cloned from the fungus and the VlPr1 protein was expressed in Escherichia coli. The VlPr1 gene contains an open reading frame (ORF) interrupted by three short introns, and encodes a protein of 379 amino acids. Protein sequence analysis revealed high homology with subtilisin serine proteases. The molecular mass of the protease was 38 kDa, and the serine protease exhibited its maximal activity at 40°C and pH 9.0. Protease activity was also affected by Mg(2+) and Ca(2+) concentration. The protease showed inhibitory activity against several plant pathogens, especially towards Fusarium moniliforme.

New insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina

BackgroundSubtilisin-like serine proteases play an important role in pathogenic fungi during the penetration and colonization of their hosts. In this study, we perform an evolutionary analysis of the subtilisin-like serine protease genes of subphylum Pezizomycotina to find if there are similar pathogenic mechanisms among the pathogenic fungi with different life styles, which utilize subtilisin-like serine proteases as virulence factors. Within Pezizomycotina, nematode-trapping fungi are unique because they capture soil nematodes using specialized trapping devices. Increasing evidence suggests subtilisin-like serine proteases from nematode-trapping fungi are involved in the penetration and digestion of nematode cuticles. Here we also conduct positive selection analysis on the subtilisin-like serine protease genes from nematode-trapping fungi.ResultsPhylogenetic analysis of 189 subtilisin-like serine protease genes from Pezizomycotina suggests five strongly-supported monophyletic clades. The subtilisin-like serine protease genes previously identified or presumed as endocellular proteases were clustered into one clade and diverged the earliest in the phylogeny. In addition, the cuticle-degrading protease genes from entomopathogenic and nematode-parasitic fungi were clustered together, indicating that they might have overlapping pathogenic mechanisms against insects and nematodes. Our experimental bioassays supported this conclusion. Interestingly, although they both function as cuticle-degrading proteases, the subtilisin-like serine protease genes from nematode-trapping fungi and nematode-parasitic fungi were not grouped together in the phylogenetic tree. Our evolutionary analysis revealed evidence for positive selection on the subtilisin-like serine protease genes of the nematode-trapping fungi.ConclusionsOur study provides new insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina. Pezizomycotina subtilisins most likely evolved from endocellular to extracellular proteases. The entomopathogenic and nematode-parasitic fungi likely share similar properties in parasitism. In addition, our data provided better understanding about the duplications and subsequent functional divergence of subtilisin-like serine protease genes in Pezizomycotina. The evidence of positive selection detected in the subtilisin-like serine protease genes of nematode-trapping fungi in the present study suggests that the subtilisin-like serine proteases may have played important roles during the evolution of pathogenicity of nematode-trapping fungi against nematodes.

The Expression of a Recombinant cry1Ac Gene with Subtilisin-Like Protease CDEP2 Gene in Acrystalliferous Bacillus thuringiensis by Red/ET Homologous Recombination

A novel cDNA encoding the subtilisin-like serine protease gene CDEP2 was isolated from Beauveria bassiana by reverse transcription polymerase chain reaction (RT-PCR). It contained an 1137 bp ORF that predicted a protein of 379 amino acids with M = 38863 Da and pI = 8.21. In an attempt to improve insecticidal activity, the CDEP2 gene and the cry1Ac gene from Bacillus thuringiensis were co-fused into the vector pHT315 as pHAc-CDEP2 plasmid by Red/ET homologous recombination. The co-fusion gene was attempted under the control of the native cry1Ac promoter. Plasmid pHAc-CDEP2 was electro-transformed into the B. thuringiensis subsp. kurstaki Cry(-)B. Analyzed by SDS-PAGE and Western blotting, the transformant Cry(-)B-pHAc-CDEP2 strain produced a 130 kDa Cry1Ac protein and 39 kDa CDEP2 protein. The 50% lethal concentration values (LC(50)) of Cry(-)B-pHAc-CDEP2 strain (8.5 microl/ml) to Helicoverpa armigera third instars larvae was clearly higher than the Cry(-)B-pHAc strain (16.7 microl/ml) at 72 h.

Isolation and Characterization of a Microsporocyte-Specific Gene, OsMSP, in Rice

A spikelet-specific cDNA clone, OsMSP (Oryza sativa meiosis serine protease), was isolated from a rice cDNA library constructed by suppression subtraction hybridization (SSH). Its expression level was significantly reduced in the thermo-sensitive genic male-sterile rice line at sterile temperature (30/26°C, day/night) compared with that at fertile temperature (23/21°C, day/night) conditions. In contrast, no significant differences were observed in the normal fertile line under the above two sets of temperature conditions. The cDNA is 2,867 bp in length with an open reading frame (ORF) encoding 837 amino acid residues. Northern blots revealed that OsMSP transcripts were specifically present in spikelets at meiosis, and reached the highest level at early microspore stage. mRNA in situ hybridization confirmed that OsMSP was a rice microsporocyte-specific gene and its expression was limited to early microspore developmental stage. BLAST and phylogenetic analyses revealed that OsMSP shared high similarities with subtilisin-like serine protease genes from other organisms at both nucleotide and amino acid levels.

Alkaliphilic Bacillus sp. strain KSM-LD1 contains a record number of subtilisin-like serine proteases genes

The presence of 11 genes encoding subtilisin-like serine proteases was demonstrated by cloning from the genome of alkaliphilic Bacillus sp. strain KSM-LD1. This strain exoproduces the oxidatively stable alkaline protease LD-1 (Saeki et al. Curr Microbiol, 47:337-340, 2003). Among the 11 genes, six genes encoding alkaline proteases (SA, SB, SC, SD, SE, and LD-1) were expressed in Bacillus hosts. However, the other five genes for subtilisin-like proteases (SF, SG, SH, SI, and SJ) were expressed in neither Bacillus hosts nor Escherichia coli. The deduced amino acid sequences of SA, SB, SC, SF, SG, SH, SI, and SJ showed similarity to those of other subtilisin-like proteases from Bacillus strains with only 38 to 86% identity. The deduced amino acid sequence of SD was completely identical to that of an oxidatively stable alkaline protease from Bacillus sp. strain SD521, and that of SE was almost identical to that of a high-molecular mass subtilisin from Bacillus sp. strain D-6 with 99.7% identity. There are four to nine subtilisin-like serine protease genes in the reported genomes of Bacillus strains. At least 11 genes for the enzymes present in the genome of Bacillus sp. strain KSM-LD1, and this is the greatest number identified to date.

Isolation of a subtilisin-like serine protease gene (MyxSubtSP) from spores of Myxobolus cerebralis, the causative agent of whirling disease

Proteases play important roles in parasite life cycles and host-parasite interactions. They are pathogenesis factors of many pathogenic organisms and are hence potential targets for chemotherapeutic treatment of disease. We identified a subtilisin-like serine protease gene, MyxSubtSP, expressed by Myxobolus cerebralis. After PCR with subtilisin-like serine protease primers, the gene was cloned, sequenced and aligned against the NCBI database. Its corresponding amino acid sequence included the putative conserved domains of Peptidase_S8, subtilase family and AprE, subtilisin-like serine proteases. Rapid amplification of 5' and 3' cDNA ends (RACE) was used to generate the full length (1385 bp) gene, with a 429 bp open reading frame. The gene encompasses coding regions for a catalytic triad formed by Asp-74, His-100 and Ser-110.

Gene from tropical Bacillus sphaericus encoding a protease closely related to subtilisins from Antarctic bacilli

We undertook to identify the protease(s) involved in the in vivo degradation of the 100 kDa mosquitocidal toxin (Mtx) from Bacillus sphaericus SSII-1 and isolated a B. sphaericus SSII-1 gene flanked upstream by a typical Shine-Dalgarno ribosome binding site and downstream by a strong ρ-independent transcription terminator. The predicted ORF encodes a 432 amino acid protein with significant homology throughout its sequence to two subtilisin-like serine proteases from the Antarctic psychrophilic (cold-adapted) bacilli, TA39 and TA41. The predicted N-terminal sequence suggests that the B. sphaericus protease is related to sfericase, a partially characterized serine protease from B. sphaericus. Only B. sphaericus strains which produce Mtx-degrading protease activity harbour the subtilisin-like protease gene, suggesting that this protease may be responsible for or contribute to the degradation of Mtx in B. sphaericus SSII-1. A 36-kDa protease with Mtx-degrading activity and similar properties to sfericase was also purified from sporulated cultures of B. sphaericus SSII-1. Further studies are needed to determine the relationship of this protease to sfericase and to the predicted product of the subtilisin-like serine protease gene. © 1997 Elsevier Science B.V. All rights reserved.